Our long term goal is to develop a feasible assay for High Throughput Screening (HTS) to identify therapeutic agents that can maximize the effectiveness of cytotoxic cancer therapies, such as radiotherapy and chemotherapy, by targeting critical DNA damage response pathways. While traditional approaches available to screen radiosensitizers and chemosensitizers are enzymatic assays for kinase inhibitors, few validated targets on protein-protein interactions have been identified for developing radiosensitizers. We have recently characterized that the NBS1-ATM interaction, a critical step to activate the ATM kinase and promote survival in response to ionizing radiation, is a novel target for radiosensitization. To identify small molecules that can inhibit the NBS1-ATM interaction, we propose to develop a Fluorescein Polarization (FP) assay utilizing an in vitro binding assay of NBS1-ATM for HTS. Aim 1 will focus on the development of the FP assay. We will generate GST-ATM fragments and Texas Redlabeled NBS1 C-terminal short peptides. Utilizing these peptides, we will test NBS1- ATM binding affinity, FP assay stability, and FP DMSO tolerance. In Aim 2 we will optimize assay parameters in the HTS setting, test the assay format with small-scale pilot screens, and develop a secondary assay for detailed evaluation of HTS hits. The research proposed in this application aims to develop a novel class of inhibitors of critical DNA damage responses with a goal to help cancer patients that do not respond well to radiotherapy. Such validated inhibitors can also be powerful tools for mechanistic studies of DNA damage response signaling. [unreadable] [unreadable] [unreadable]